Microorganisms such as filamentous fungi, yeast and algae produce a variety of lipids, including fatty acyls, glycerolipids, phospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids and prenol lipids. One class of lipids commonly extracted from microbes is glycerolipids, including the fatty acid esters of glycerol (“triacylglycerols” or “TAGs”). TAGs are the primary storage unit for fatty acids, and thus may contain long chain polyunsaturated fatty acids (PUFAs), as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. There has been growing interest in including PUFAs, such as eicosapentaenoic acid [“EPA”; omega-3] and docosahexaenoic acid [“DHA”; omega-3], in pharmaceutical and dietary products. Means to efficiently and cost-effectively produce lipid compositions comprising PUFAs are therefore particularly desirable.
There are a variety of commercial sources of PUFAs. However, there are several disadvantages associated with these methods of production using natural sources. First, natural sources, such as fish and plants, tend to have highly heterogeneous oil compositions. The oils obtained from these sources therefore can require extensive purification to separate or enrich one or more of the desired PUFAs. Fish oils commonly have unpleasant tastes and odors, which may be impossible to separate economically from the desired product and can render such products unacceptable as food supplements. Unpleasant tastes and odors can make medical regimens based on ingestion of high dosages undesirable, and may inhibit compliance by the patient.
Fish may accumulate environmental pollutants and ingestion of fish oil capsules as a dietary supplement may result in ingestion of undesired contaminants. Natural sources of PUFAs are also subject to uncontrollable fluctuations in availability (e.g., due to weather, disease, or over-fishing in the case of fish stocks). Also, crops that produce PUFAs often are not competitive economically with hybrid crops developed for food production. Large-scale fermentation of some organisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella) can also be expensive and/or difficult to cultivate on a commercial scale. As a result of these limitations, extensive work has been conducted toward the development of recombinant oleaginous microorganisms that can produce PUFAs efficiently and economically at a commercial scale (e.g., U.S. Pat. Appl. Publ. No. 2005-0136519-A1). Additionally, the modification of fatty acid biosynthetic pathways in recombinant oleaginous microorganisms to enable production of desired PUFAs has also been reported (e.g., U.S. Pat. Appl. Publ. Nos. 2006-0110806-A1, 2006-0115881-A1, 2009-0093543-A1, and 2010-0317072-A1). However, there is still a need for recombinant oleaginous microorganisms having increased oil content relative to the oil of currently known strains.
U.S. Pat. No. 7,256,014 discloses that the expression of at least one plant oleosin gene in a microbial cell engineered to produce a hydrophobic/lipophilic compound, such as a carotenoid, significantly increases the overall titer of the compound.
Froissard et al. (FEMS Yeast Res. 9:428-438, 2009) disclose that the non-oleaginous yeast, Saccharomyces cerevisiae, transformed with a heterologous gene encoding a caleosin polypeptide (Arabidopsis thaliana caleosin 1, AtClo1), exhibited an increase in the number and size of lipid bodies and accumulated more fatty acids than the parent strain.
However, there are no reports of recombinant oleaginous microorganisms transformed with a gene encoding a caleosin polypeptide to increase the oil content of such recombinant microbial cells.